Bone marrow-derived mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into multiple cell lineages. Because of these capabilities, MSCs play an important role in continuous maintenance and repair of most tissue types. The quantity and quality of MSCs decrease with aging, which, in turn, is associated with the progressive failure of function of tissues and organs. Recently, we reported in mice that defects in the self-renewal and bone formation capacity of aged MSCs were completely restored by exposure to extracellular matrix (ECM) made by marrow stromal cells from young animals, and such improvement was substantially diminished when cells (from either young or old mice) were cultured on ECM made by old marrow stromal cells. This led us to investigate whether this phenomenon also occurs in humans. Indeed, our preliminary studies in human subjects clearly suggest that culturing aged MSCs on ECM made by young marrow stromal cells is able to improve their number and quality. The goal of this proposal is to determine the mechanisms whereby young ECM rejuvenates old bone marrow-derived MSCs. A unique, cell-free ECM model will be used to dissect how young ECM restores the functions of old MSCs in vitro and in vivo. The hypothesis proposed in this application is that age negatively impacts the formation of an ECM that normally preserves MSC function, and the quantity and quality of aged MSCs can be improved by exposure to an ECM made by stromal cells from young donors. We are uniquely-positioned to test this hypothesis in humans since, in addition to our strong preliminary data, up to 100 human bone marrow samples have been accumulated in the past three years from elders (60 to 96 years old). To test this hypothesis, we will systematically evaluate biological activities in viro and in vivo of young vs. old MSCs maintained on young- or old-ECM (Specific aim 1), and conduct proteomic analysis of young- and old-MSCs exposed to young- or old-ECM to elucidate the underlying changes at the protein level that are associated with the restored functions of the rescued cells (Specific aim 2). To fully understand the mechanisms underlying the influence of MSC behavior, we will determine the differences in the structures of young- vs. old-ECM including the architecture, mechanical properties, and protein composition (Specific aim 3). These studies are innovative in that we will have established, for the first time, a unique in vitr human model to examine effects of age on MSCs (intrinsic theory), and changes to MSCs by the surrounding ECM (extrinsic theory). Our studies are important from a translational point of view because they will provide key information that can facilitate the use of a patient's own (autologous) stem cells for cell- based therapies. In view of the fact that the elderly are the mai target population for this type of treatment, understating age-related changes in both MSCs and host is essential for improving the clinical outcome of transplanted autologous MSCs in old veteran.